Maria Pennuto - Academia.edu (original) (raw)

Papers by Maria Pennuto

Expansion of polyglutamine in the gene coding for androgen receptor (AR) is responsible for the d... more Expansion of polyglutamine in the gene coding for androgen receptor (AR) is responsible for the degeneration and loss of lower motor neurons in spinal and bulbar muscular atrophy (SBMA). SBMA is one out of nine neurological conditions caused by expanded polyglutamine. Recent evidence suggests the involvement of native protein structure and function in the pathogenesis of polyglutamine diseases. However, the details of how protein context affects disease pathogenesis are poorly understood. We identified protein arginine methyltransferase 6 (PRMT6) as a novel co-activator of normal and mutant androgen receptor, whose function is enhanced by polyglutamine expansion. The interaction of PRMT6 with mutant androgen receptor requires the steroid receptor interaction motif, LXXLL, of PRMT6 and the activating function 2 (AF-2) of androgen receptor. The catalytic activity of PRMT6 is also required. Consistent with this, we show that androgen receptor is a substrate of PRMT6. PRMT6 methylates the androgen receptor at the arginine residues spanning the Akt consensus sites RXRXXS. Importantly, arginine methylation by PRMT6 and serine phosphorylation by Akt at these sites is mutually exclusive. Knock down of PRMT6 in flies attenuates the degenerative phenotype caused by mutant androgen receptor. Collectively, these data provide insights into the mechanism through which protein structure and function cooperate to cause neurodegeneration and implicate arginine methylation and PRMT function in the pathogenesis of polyglutamine diseases

Scientific Reports, Sep 28, 2020

Mutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposi... more Mutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1 α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein-protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). We found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoformspecific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already shown for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell metabolic activity and necrosis, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data show an isoformspecific interaction of pVHL with MDM2, suggesting an interplay between these two E3 ubiquitin ligases. Oxygen availability regulates a plethora of physiological processes, such as cell metabolism, mitochondrial ATP production, embryo development and inflammation, and it plays a prominent role in the pathophysiology of cancer 1-3. Hypoxia response in cells is carried out by the hypoxia-inducible factor (HIF) complex in a tightly oxygen-dependent fashion 4,5. HIF is a heterodimeric complex formed by an α-subunit, whose stability is finely regulated by the intra-cellular oxygen concentration, and a constitutively expressed nuclear β-subunit 6,7. As a transcription factor, HIF is a master activator of many genes that allow the cells to cope with low oxygen growth conditions by enhancing glycolytic flux while inhibiting mitochondrial respiration 8. In physiological conditions, the levels of HIF-1α are regulated by the von Hippel-Lindau (pVHL) tumor suppressor, which continuously targets HIF-1α for proteasomal degradation. As a substrate recognition particle of the VCB multiprotein complex (together with Elongin B, Elongin C, the E3-ubiquitin ligase Cullin-2 and RBX1 9), pVHL recognizes two proline residues of HIF-1α 10 upon hydroxylation by prolyl hydroxylase domain containing proteins (PHD) 11 , whose expression in turn is closely regulated by HIF-1α itself 12. In hypoxia, the activity of PHDs is inhibited, thus resulting in HIF-1α stabilization and activation of the hypoxia-response gene expression program. Together, pVHL, HIF-1α and PHD form the cellular oxygen-sensing system 4,11,13,14. Deregulation of HIF-1α/pVHL promotes sustained angiogenesis 15 and is causative of von Hippel-Lindau syndrome 16-18 , a familiar predisposition to develop different types of cancer, such as pheochromocytoma, paraganglioma, retinal-and cerebellar-hemangioblastoma, and renal cell carcinoma (RCC) 19. In humans, the VHL gene is located on chromosome 3 20. Three biologically active pVHL isoforms have been described so far, namely pVHL30, pVHL19, and pVHL172. Both pVHL19 and pVHL30 act as tumor suppressors and have a redundant function to promote HIF-1α degradation, yet they have a different sub-cellular localization 21 and isoform-specific

Androgen receptor (AR) is a cytoplasmic transcription factor whose function is regulated by testo... more Androgen receptor (AR) is a cytoplasmic transcription factor whose function is regulated by testosterone/dihydrotestosterone (T/DHT). PolyQ expansion in the AR gene causes SBMA, a neuromuscular disease triggered by hormone binding to AR. S-palmitoylation is a post-translational modification referring to the covalent attachment of palmitate moieties to cysteine residues. Regulated cycles of palmitoylation and depalmitoylation lead to reversible membrane association. This study aims to explore the regulatory mechanism of AR palmitoylation and its potential implications in SBMA. By cDNA transfection, membrane fractionation and confocal microscope analysis performed in HEK 293T cells and motor neuron derived MN1cells, we present evidence that both wild-type AR24Q and mutant AR65Q are in part localized to intracellular membranes and that membrane binding occurs uniquely through palmitoylation. In silico analysis revealed several predicted palmitoylation sites on AR, in addition to the previously described cysteine 807. We performed site-directed mutagenesis to abolish putative AR palmitoylation sites, changing cysteine 577, 580 and 615 into serines and are now evaluating which mutants lose membrane-binding and 3H-palmitate incorporation abilities. AR phosphorylation at serines 215 and 792 by Akt has been shown to reduce polyQ-AR toxicity by impairing AR nuclear translocation, due to acquired DHT treatment resistance. Interestingly, we show that AR molecules bearing Ser215Asp and Ser792Asp mutations, where aspartate mimics constitutive AR phosphorylation, are entirely recovered in membrane fractions, suggesting a correlation between phosphorylation by Akt and AR palmitoylation. We propose reversible palmitoylation as a way to control polyQ-expanded AR function by down-modulating its nuclear translocation and therefore reducing gene transactivation

The neuromuscular disease Spinal bulbar muscular atrophy (SBMA) associates with loss of bulbar or... more The neuromuscular disease Spinal bulbar muscular atrophy (SBMA) associates with loss of bulbar or spinal motoneurons and skeletal muscle atrophy. SBMA is caused by a mutation of the androgen receptor (AR) gene resulting in a protein with an elongated polyglutamine (polyQ) tract. ARpolyQ acquires nuclear toxicity after binding testosterone, which induces AR nuclear translocation and ARpolyQ misfolding. Misfolded ARpolyQ is prone to aggregate, a process counteracted by the protein quality control (PQC) system. This system comprises chaperones and the degradative pathways (proteasome and autophagy). Several data suggest that misfolded ARpolyQ is mainly process via autophagy, and causes autophagy flux blockage. Restoration of a functional autophagy is beneficial to cells expressing misfolded ARpolyQ. A peculiar form of autophagy is the "chaperone-assisted selective autoghagy" (CASA), which relies on dynein-mediated retrograde transport of the CASA (HSPB8-BAG3-HSC70-CHIP) compl...

Some familial forms of Amyotrophic Lateral Sclerosis (ALS) are characterized by a dominant mutati... more Some familial forms of Amyotrophic Lateral Sclerosis (ALS) are characterized by a dominant mutation in superoxide dismutase (SOD) 1 gene. Many evidence suggested that SOD1 toxicity is non-cell autonomous, involving multiple cell types: affected motor neurons, glial cells and muscle cells. In particular, muscle might be a primary source of toxicity. Indeed, it is reported that in mice the expression of mutated SOD1 in muscle cells is sufficient to induce motor neuron degeneration and muscle abnormalities already at the pre-symptomatic stage. TGFbeta1 is a growth factor known to be involved in neuron survival and in muscle development/maintenance. Furthermore, TGFbeta1 levels are increased in serum of ALS patients. On these bases, we decided to evaluate the expression of the TGFbeta1 gene in skeletal muscle and spinal cord of transgenic mice expressing mutated hSOD1 at different stages of the disease and taking in consideration mouse sex. The results indicate that in muscle TGFbeta1 expression is up-regulated by mutated hSOD1 at the symptomatic stage both in male and female, while at the pre-symptomatic stage TGFbeta1 mRNA levels are increased only in male. An high expression of TGFbeta1 might be responsible of muscle fibrosis. In spinal cord, the expression of TGFbeta1 decreases in the pre-symptomatic mice, indicating a drop in motorneuron protection. On the contrary, TGFbeta1 mRNA levels increase at the symptomatic stage, probably for the participation of other cell types. The analysis of the expression of Smad proteins, which are the main signalling molecules activated by TGFbeta, indicates a different regulation in muscle and spinal cord, and between male and female mice. Grants: ARISLA, Telethon-Italy, Fondazione Cariplo, AFM

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal... more Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a CAG triplet repeat in exon 1 of the androgen receptor (AR) gene. This expansion is translated into an abnormally long polyglutamine tract (polyQ) in the AR protein. The AR is a transcription factor that mediated the biological activities of male sexual hormones. Several evidence suggests that the elongated polyQ tract confers a toxic gain-of-function to the mutant AR. This ARpolyQ toxicity is activated by the AR ligand testosterone (or dihydrotestosterone). Testosterone-dependent toxicity is triggers by the generation of ARpolyQ misfolded species, that in some conditions, tend to aggregate in spinal cord motoneurons and muscle cells. ARpolyQ aggregates are formed in the cytoplasm and nuclei of affected cells, but they toxic role is still largely debated. Instead, there is general agreement on the fact that ARpolyQ toxicity is associated with the nuclear localization of an altered (misfolded) fraction of the protein. Therefore, the prevention of nuclear localization and/or the facilitation of ARpolyQ misfolded fraction degradation may have great beneficial effects against ARpolyQ toxicity. Recently, we demonstrated that the ubiquitin proteasome system (UPS) degrades a large fraction of misfolded ARpolyQ. However, an excess of ARpolyQ may overwhelm the UPS, escaping the degradation and thus tend to accumulate in aggregates. These aggregates are useful to confine the toxic protein in a physically defined subcellular compartment, thus reducing the potential toxicity of undegraded misfolded ARpolyQ. Generally, the aggregates are transported to the microtubule organization center (MTOC) to be engulfed into autophagosomes and destroyed by the autophagic system. When an insufficient pool of specific chaperones is present, the autophagic flux may be blocked and this lead to the formation of insoluble inclusions. In fact, we found that the silencing of HspB8, a member of the small heat shock protein family, correlated with a massive accumulation of misfolded ARpolyQ in immortalized motoneuronal cells. Conversely, HspB8 overexpression facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. HspB8 is highly induced in surviving motoneurons of patients affected by motoneuron diseases and HspB8 participates in the stress response aimed at cell protection. HspB8 does not induce autophagy (p62 and LC3 expression, two key autophagic molecules), but it prevents p62 bodies formation, restoring a fully functional autophagic flux. Notably, we found that, trehalose, a natural disaccharide know to be a potent autophagy stimulator, induces HspB8 expression suggesting that HspB8 could be one of the molecular mediators of the pro-autophagic activity of trehalose. Therefore, based on the evidence that testosterone triggers nuclear toxicity by inducing AR nuclear translocation, and misfolded species accumulate because of an impaired autophagic flux, we hypothesize that prevention of ARpolyQ nuclear localization, combined with an increased ARpolyQ cytoplasmic clearance should reduce its toxicity. We thus used the antiandrogen Bicalutamide (Casodex\uae), which slows down AR activation and nuclear translocation, in combination with trehalose, and we found that in motoneurons the two compounds synergically reduced ARpolyQ insoluble forms, with an efficiency higher than the one observed in single treatments. This effect was also present on insoluble species of AR with a very long polyQ (Q112) tract, capable to generate nuclear aggregates into the cell nuclei. Therefore, the combinatory use of Bicalutamide and trehalose greatly facilitate ARpolyQ clearance, and might be a promising approach to be tested in vivo in animal models of SBMA

Frontiers in Endocrinology, 2019

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mut... more Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mutant androgen receptor (AR) containing an elongated polyglutamine (polyQ) tract. ARpolyQ toxicity is triggered by androgenic AR ligands, which induce aberrant conformations (misfolding) of the ARpolyQ protein that aggregates. Misfolded proteins perturb the protein quality control (PQC) system leading to cell dysfunction and death. Spinal cord motoneurons, dorsal root ganglia neurons and skeletal muscle cells are affected by ARpolyQ toxicity. Here, we found that, in stabilized skeletal myoblasts (s-myoblasts), ARpolyQ formed testosterone-inducible aggregates resistant to NP-40 solubilization; these aggregates did not affect s-myoblasts survival or viability. Both wild type AR and ARpolyQ were processed via proteasome, but ARpolyQ triggered (and it was also cleared via) autophagy. ARpolyQ reduced two pro-autophagic proteins expression (BAG3 and VCP), leading to decreased autophagic response in ARpolyQ s-myoblasts. Overexpression of two components of the chaperone assisted selective autophagy (CASA) complex (BAG3 and HSPB8), enhanced ARpolyQ clearance, while the treatment with the mTOR independent autophagy activator trehalose induced complete ARpolyQ degradation. Thus, trehalose has beneficial effects in SBMA skeletal muscle models even when autophagy is impaired, possibly by stimulating CASA to assist the removal of ARpolyQ misfolded species/aggregates.

Neurobiology of Aging, 2019

Transforming Growth Factor beta 1 signaling is altered in the spinal cord and muscle of amyotroph... more Transforming Growth Factor beta 1 signaling is altered in the spinal cord and muscle of amyotrophic lateral sclerosis mice and patients.

European Journal of Neurology, 2016

Disease severity varies considerably among patients with Spinal and Bulbar Muscular Atrophy (SBMA... more Disease severity varies considerably among patients with Spinal and Bulbar Muscular Atrophy (SBMA). Our aim was to investigate the role of androgen receptor (AR) polymorphic repeats in SBMA phenotype. We analyzed the length of AR polyQ and polyG tracts in 159 SBMA patients. No relationship between polyG size or polyG/polyQ haplotypes and clinical phenotype was found. An independent negative correlation between polyQ-length and onset of weakness was confirmed (P < 0.001). The negative results of our study prompt to continue the search for potential disease modifiers in SBMA outside the AR gene.

Neurology, Apr 6, 2015

OBJECTIVE: To describe the clinical phenotype in a large population of Kennedy’s disease (KD) pat... more OBJECTIVE: To describe the clinical phenotype in a large population of Kennedy’s disease (KD) patients. BACKGROUND: KD is a neuromuscular disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. AR is broadly expressed in the body, thus AR toxicity may involve organs other than the neuromuscular system. METHODS: A clinical protocol focused on androgen-linked functions was administered to 72 Caucasian KD patients. It included: neurological exam, blood tests (glucose and lipid metabolism, hormonal status, bone metabolism), bone density test, and EKG. Urinary and sexual functions were also assessed by the means of functional scores and prostate echography. RESULTS: Mean age at onset was 46 years and lower limb weakness was reported as presenting symptom in most cases. Patients presented a tendency to overweight with sugar levels consistent with diabetes mellitus or with impaired glucose tolerance in almost 50[percnt] of cases. Thirty-eight per cent of patients had borderline blood total cholesterol levels, while 15[percnt] had an overt hyperlipidemia. Hormonal assessment was unremarkable. Bone density test showed femoral osteopenia in 45[percnt] of patients regardless of their age. No significant heart rhythm disorders were recorded. One third (37[percnt]) of patients complained urinary symptoms, which scored moderate to severe according to the functional scales and were unrelated to prostate hypertrophy. Erectile dysfunction was reported in 46[percnt] of cases. CONCLUSIONS: This study further widens the clinical spectrum of KD. Disclosure: Dr. Querin has nothing to disclose. Dr. Bertolin has nothing to disclose. Dr. Volpe has nothing to disclose. Dr. Zara has nothing to disclose. Dr. Da Re has nothing to disclose. Dr. Zoccarato has nothing to disclose. Dr. Caretta has nothing to disclose. Dr. Foresta has nothing to disclose. Dr. Marcato has nothing to disclose. Dr. Iafrate has nothing to disclose. Dr. Corrado has nothing to disclose. Dr. Silvano has nothing to disclose. Dr. Pegoraro has received personal compensation for activities with BioMarin Pharmaceutical Inc., and MEDA Pharmaceuticals Inc. as a scientific advisory board member and/or consultant. Dr. Pareyson has nothing to disclose. Dr. Pennuto has nothing to disclose. Dr. Soraru has nothing to disclose.

European Psychiatry, 2014

Major depressive disorder (MDD) is characterized by alterations of brain function that are identi... more Major depressive disorder (MDD) is characterized by alterations of brain function that are identifiable also during the brain's 'resting state'. One functional network that is disrupted in this disorder is the default mode network (DMN), a set of large-scale connected brain regions that oscillate with low-frequency fluctuations and are more active during rest relative to during a goal-directed task. Recent studies support the idea that the DMN is not a unitary system, but rather is composed of smaller and distinct functional subsystems that interact with each other. The functional relevance of these subsystems in depression, however, is unclear. Here, we investigate the functional connectivity of distinct DMN subsystems and their interplay in depression using resting state functional magnetic resonance imaging (rs-fMRI). We show that patients with MDD exhibit increased within-network connectivity in posterior, ventral and core DMN subsystems along with reduced interplay from the anterior to the ventral DMN subsystems. These data suggest that MDD is characterized by alterations of subsystems within the DMN as well as their interactions. Our findings highlight the critical role of DMN circuitry in the pathophysiology of MDD, thus suggesting these subsystems as potential therapeutic targets.